The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants and to transforming genes that enhance disease resistance.
Disease in plants is produced by biotic and abiotic causes. Biotic causes include fungi, viruses, insects, bacteria, and nematodes. Of these, fungi are the most frequent causative agents of disease in plants. Abiotic causes of disease in plants include extremes of temperature, water, oxygen, soil pH, plus nutrient-element deficiencies and imbalances, excess heavy metals, and air pollution.
Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack.
A host of cellular processes enables plants to defend themselves from disease caused by pathogenic agents. These processes apparently form an integrated set of resistance mechanisms that is activated by initial infection and then limits further spread of the invading pathogenic microorganism.
After recognition of a potentially pathogenic microbe, plants can activate an array of biochemical responses. Generally, the plant responds by inducing several local responses in the cells immediately surrounding the infection site. The most common resistance response observed in both nonhost and race-specific interactions is termed the xe2x80x9chypersensitive responsexe2x80x9d (HR). In the hypersensitive response, cells contacted by the pathogen, and often neighboring cells, rapidly collapse and dry producing a necrotic fleck. Other responses include the deposition of callose, the physical thickening of cell walls by lignification, and the synthesis of various antibiotic small molecules and proteins. Genetic factors in both the host and the pathogen determine the specificity of these local responses, which can be very effective in limiting the spread of infection.
In many plant species, an initial inoculation by a necrotizing pathogen can immunize the plant to subsequence infection. Particularly well characterized examples of plant immunity are the phenomenon of systemic acquired resistance (SAR) and induced resistance. In these systems, inoculation by a necrotizing pathogen results in systemic protection against subsequent infections by that pathogen as well as a number of other agronomically important bacterial, fungal and viral pathogens. Systemic acquired resistance can also be triggered by chemical immunization.
SAR is characterized by the expression of SAR genes, including pathogenesis-related (PR) genes. The SAR genes are induced following infection by a pathogen. Some of these genes have a role in providing systemic acquired resistance to the plant. The resulting proteins are believed to be a common defensive systemic response of plants to infection by pathogens. When the SAR signal transduction pathway is blocked, plants become more susceptible to pathogens that normally cause disease, and they become susceptible to some infectious agents that would not normally cause disease. Salicylic. acid (SA) accumulation appears to be required for SAR signal transduction.
A gene in Arabidopsis identified as NPRI or NIM1 has recently been found which controls the onset of SAR (Cao, et al., Cell, 88:57-63 (1997); WO 97/49822; and WO 98/826082, all of which are herein incorporated by reference). A mutation in the NPR1 gene (nim1/sai1) results in enhanced disease susceptibility (Cao, et al., The Plant Cell, 6:1583-1592 (1994); Volko, et al., Genetics, 149:537-548; Shah, et al., MPMI, 10(1):69-78 (1997); and Delaney, et al., Proc. Natl. Acad. Sci. USA, 92:6602-6606 (1995)). By overexpression of the NPR1 gene in Arabidopsis a battery of downstream pathogenesis-related genes are induced (Cao, et al., Proc. Natl. Acad. Sci. USA, 95:6531-6536 (1998). This overexpression of NPR1 conferred resistance to the pathogens Pseudomonas syringae and Peronospora parasitica with no obvious detrimental effects on the plants.
Expression of heterologous DNA sequence in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Thus, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in expression constructs of transformation vectors to bring about varying levels of constitutive expression of heterologous nucleotide sequence in a transgenic plant.
Frequently it is desirable to have constitutive expression of a DNA sequence throughout the cells of an organism. For example, increasing resistance of a plant to infection by soil- and air-borne pathogens might be accomplished by genetic manipulation of the plant""s genome to comprise a constitutive promoter operably linked to a heterologous pathogen-resistance gene such that pathogen-resistance proteins are continuously expressed throughout the plant""s tissues.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant""s tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a constitutive promoter operably linked to an antisense nucleotide sequence, such that constitutive expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
Thus, isolation and characterization of constitutive promoters that can serve as regulatory regions for constitutive expression of heterologous nucleotide sequences of interest are needed for genetic manipulation of plants to exhibit specific phenotypic traits.
In the present invention, a new NPR1 polynucleotide isolated from maize is disclosed. By manipulation of the NPR1 polynucleotide in maize or in other plants, the plant can become resistant to a number of plant pathogens. The present invention provides a new method of conferring disease resistance to plants. In addition, the present invention provides promoter regulatory elements associated with the maize NPR1 polynucleotide that confer constitutive expression on a heterologous polynucleotide and are also capable of inducing expression of the heterologous polynucleotide to a higher level in the presence of a pathogen or fungal elicitor. Further, the present invention describes a method for evaluating putative activators of the plant defense pathway.
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to NPR1. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention. Another object of the present invention it to provide promoters capable of driving expression in a constitutive manner, which are also inducible in the presence of a pathogen or fungal elicitor. An additional object of the present invention is to provide methods for screening putative activators of a plant resistance pathway.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide amplified from a Zea mays nucleic acid library using the primers of the present invention; (c) a polynucleotide comprising at least 20 contiguous bases of the polynucleotides of the present invention; (d) a polynucleotide encoding a maize NPR1 protein; (e) a polynucleotide having at least 70% sequence identity to the polynucleotides of the present invention; (f) a polynucleotide comprising at least 25 nucleotides in length which hybridizes under low stringency conditions to the polynucleotides of the present invention; (g) a polynucleotide comprising the sequence set forth in SEQ ID NOS: 1 or 3; and (f) a polynucleotide complementary to a polynucleotide of (a) through (g). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette with a promoter operably linked to any of the isolated nucleic acids of the present invention. Preferred plants containing the recombinant expression cassette of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. The present invention also provides transgenic seed from the transgenic plant.
In another aspect, the present invention relates to an isolated protein selected from the group consisting of (a) a polypeptide comprising at least 25 contiguous amino acids of SEQ ID NO: 2 or 4; (b) a polypeptide which is a maize NPR1; (c) a polypeptide comprising at least 70% sequence identity to SEQ ID NO: 2 or 4; (d) a polypeptide encoded by a nucleic acid of the present invention; and (e) a polypeptide characterized by SEQ ID NO: 2 or 4.
In a further aspect, the present invention relates to a method of modulating the level of protein in a plant by introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; culturing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to modulate the protein of the present invention in the plant. Preferred plants of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. The level of protein in the plant can either be increased or decreased.
The present invention also provides for an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid driving expression of a gene for a maize NPR1; b) a nucleic acid comprising at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 5; c) a nucleic acid having at least 70% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 5; d) a nucleic acid set forth in SEQ ID NO: 5; and e) a nucleic acid that hybridizes to any of a), b), c), or d) under stringent conditions. The present invention also includes recombinant expression cassettes, vectors, host cells including plant cells, plants and seeds containing the previously described nucleic acid molecule capable of initiating constitutive transcription in a plant cell.
Additionally, the present invention provides for a method for constitutively expressing a heterologous nucleic acid in a plant, comprising introducing into a plant cell or tissue a promoter of the present invention; and culturing the transgenic plant cell or tissue under plant growing conditions to produce a regenerated plant.
In another aspect, the present invention provides for a method of conferring disease resistance to a plant, the method comprising introducing into a plant cell or tissue a vector comprising a maize NPR1 polynucleotide operably linked to a promoter; culturing the transgenic plant cell or tissue under plant growing conditions to produce a regenerated plant; and inducing expression of the maize NPR1 polynucleotide for a time sufficient to confer disease resistance to a plant.
The present invention also provides for a method for screening putative activators of a plant resistance pathway comprising selecting an putative activator gene to test; introducing a plant cell or tissue with an expression cassette containing the putative activator gene operably linked to a constitutive promoter and an expression cassette containing a scorable marker gene operably linked to an inducible promoter; and scoring the plant cell or tissue to determine if expression of the scorable marker gene has increased. The scorable marker gene is preferably selected from beta-glucuronidase, luciferase, anthocyanin pathway transcriptional activators, and green florescent protein. The inducible promoter may be a wound inducible promoter, a stress inducible promoter, or preferably a pathogen inducible promoter. The method of introducing the expression cassette is optionally by ballistic particle acceleration or by Agrobacterium-mediated delivery.